Imbalances in the essential mineral elements copper, magnesium and zinc have been demonstrated in a number of immunologically related diseases. Several groups have reported on the role of zinc in T-cell functions and specifically the effects of zinc deficiency on T helper cell activity in long-term in vivo dietary zinc deficiency models. Problems in interpreting these data have developed since immune cells were cultured in zinc containing media when the assays for immunological reactivity were done. We developed an in vitro culture method that permits us to control the elemental environment of the immune cell populations. Our goal is to study specific mechanisms dependent on the three minerals in the generation of cytotoxic T lymphocytes (CTL). To accomplish this aim, we use in vitro alloantigen-stimulated mixed lymphocyte cultures as we study the involvement of copper, magnesium and zinc in three phases of CTL generation: the initiation, proliferation and lytic phases. Studies have focused on determining at what step and at what level of mineral deficiency CTL generation is modified by the lack of metals. Results from experiments on the initiation and proliferation phases indicate that relatively high levels of the three minerals are needed (greater than 70% of control values). Second, we are comparing the changes in CTL generation of splenocytes from mice dietarily made deficient in copper, magnesium and zinc with splenocytes that have been made deficient by in vitro methods. These comparisons are necessary to ascertain the impact of metal deficiencies and what role in vitro culture has on the reactivity of T cells. We have added studies on the function of accessory cells and have been initially looking at Ia expression by in vitro deficient cells. Scanning and electron micrographs of in vitro deficient T cells and macrophages have shown significant changes in cell size and surface configuration. Last, we are studying the effects of deficiencies of copper, magnesium and zinc on the synthesis of the "helper" factors interleukin-1 and interleukin-2, both of which are required for CTL generation, and have found production of both factors, but with time shifts as to when in the cultures they are produced.